B lymphocytes are white blood cells that provide immunological protection against foreign proteins, including infections. However, it is increasingly being recognized that an aberration in immunological responses by B cells may also play an important role in human inflammatory disorders. One such disorder is multiple sclerosis (MS), which is the most common inflammatory disorder of the brain and spinal cord in humans, and which is the second most common cause of neurological disability in adults. Recent epidemiological assessments suggest that over 28,000 Veterans with multiple sclerosis (MS) receive medical care annually in VA (http://www.va.gov/health/NewsFeatures/20110309a.asp). World-wide, there are 2.5 million physician-diagnosed patients with MS. Perhaps the best evidence to date that support a role for B-cell involvement in MS pathogenesis come from clinical trials with rituximab in MS. Rituximab is a pharmacological agent that depletes B lymphocytes in the blood and the brain. Data in this regard was published by my laboratory and is shown in this grant proposal. Despite the recognition that rituximab benefits patients with MS, many questions regarding B cells in this disorder have remained unanswered. Importantly, it is currently unclear whether B cells are required in the brain and spinal cord to contribute to MS disease burden, or whether their role is limited to organs outside the central nervous system. While B cells may be depleted by rituximab, it is unlikely that these agents affect all B cells that are already in the brain and spnal cord during later stages of the disease. Other recent observations by other investigators and our group suggest that not all B cells in MS are contributing to disease. Some B cells appear to be diminishing disease activity. We refer to these B cells as B regulatory cells, and to all other B cells as B bon-regulatory cells. My laboratory recent has recently generated mice in which B cells cannot migrate into the brain and spinal cord. These animals are otherwise developmentally normal. The generation of these CD19.Cre+/--4-integrinf/f mice provides us a unique opportunity to clarify the role of B cells in compartments outside of the central nervous system and within it. Based on the published work by other investigators, we are also now able to generate different kinds of B regulatory cells and B non-regulatory cells. Based on our preliminary results, and using the reagents my laboratory has generated, we will test three hypothesis regarding the role of B cell subsets in the experimental autoimmune encephalomyelitis (EAE) animal model of MS: a. we hypothesize that the generation of B regulatory cells outside of the brain and spinal cord is required for amelioration of early stages of CNS autoimmune disease; b. we hypothesize that migration of B non-regulatory cells into the brain and spinal cord is critical in perpetuating EAE clinical disease activity; Ideally, one correlates observations made in an animal model with events that occur in patients. This can be challenging, as patients often take different forms of treatment that can affect immunological parameters, including the number and type of cells in different compartments of the body. To circumvent these sources of error, we will conduct mechanistic studies during a phase II multi-national randomized double-blind clinical trial that was initiated and is headed by the PI of this grant proposal. All clinical and imaging studies of this grant are funded by a separate entity, and are not subject of this application. The clinical trial will assess patients who have been treated with the FDA-approved agent natalizumab. Natalizumab prevents cells, including B cells, from migrating into the brain and spinal cord. Natalizumab will be discontinued in all patients, and they will be started on another FDA-approved therapy, glatiramer acetate (GA), which induces B regulatory cells. Based on our preliminary animal and human data, c. we hypothesize that the FDA-approved agent natalizumab prevents the migration of B regulatory cells and B non-regulatory cells into the CNS; d. we hypothesize that the FDA-approved agent GA promotes the generation of B regulatory cells in the periphery of patients with MS, which eventually will gain access to the CNS. We are confident that our studies will substantially increase our knowledge of B cell subsets as disease modifiers and as potential therapeutic agents in patients with MS.